Diagnostic system for hemoglobin analysis

ABSTRACT

A diagnostic system detects and/or measures hemoglobin variants in blood of subject, such as HbA1c, to determine blood glucose concentration in the subject.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/800,137, filed Feb. 1, 2019, the subject matter of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention is related to a diagnostic system, andparticularly relates to a diagnostic system that includes anelectrophoresis device that rapidly and easily perform blood analysis.

BACKGROUND

Monitoring of the course of diabetes in a patient may be accomplished bychecking the glucose level in the blood. However, changes in this levelare known to be especially rapid. Glucose assays can give only sporadicinformation about the patient's blood sugar level, and hence do notreflect the changes in the latter in the weeks preceding the analysis.

Quantitative determination of glycosylated hemoglobin A1 (HbA1c) isknown to reflect a patient's average blood glucose concentration over aperiod of two months preceding the taking of a blood sample. HbA1c isdefined by the International Federation of Clinical Chemistry workinggroup (IFCC) as hemoglobin that is irreversibly glycated at one or bothN-terminal valines of the beta chains. It is formed from irreversible,slow, non-enzymatic addition of a sugar residue to the hemoglobin, andthe rate of production is directly proportional to the ambient glucoseconcentration. The long lifespan of erythrocytes (mean 120 days) enablesHbA1c to be used as an index of glycemic control over the preceding twoto three months and as the adequacy of treatment in diabetic patients.For this reason, HbA1c is widely used in a screening test for diabetesmellitus and as a test item for checking whether a diabetic keeps theblood sugar under control.

Conventionally, HbA1c has been measured by HPLC, immunoassay,electrophoresis or the like. HPLC is widely used in clinicalexaminations. HPLC requires only 1 to 2 minutes to measure each sample,and has achieved a measurement accuracy of about 1.0% in terms of a CVvalue obtained by a within-run reproducibility test. Measurement methodsfor checking whether a diabetic keeps the blood sugar under control arerequired to perform at this level.

Measurement of hemoglobin by electrophoresis has been used for a longtime to separate abnormal Hbs with an unusual amino acid sequence.However, separation of HbA1c is significantly difficult, and takes 30minutes or more by gel electrophoresis. Thus, electrophoresis has beenunsatisfactory in terms of measurement time and measurement accuracywhen applied to the clinical examinations. Therefore, electrophoresishas hardly been applied to clinical diagnosis of diabetes.

SUMMARY

Embodiments described herein relate to a diagnostic system andelectrophoresis device for detecting and/or measuring hemoglobinvariants in blood of subject, and particularly relates to a cartridge ofan electrophoresis device for a point-of-care diagnostic system formeasuring hemoglobin (Hb) types, such as HbA1c, in a subject todetermine blood glucose concentration in the subject. In someembodiments, the diagnostic system can be used to measure HbA1c levelsto determine glucose levels in a subject having or suspected of havingdiabetes.

In some embodiments, the cartridge includes a housing having amicrochannel structured to receive hemolysate of a blood sample. Themicrochannel extends between first and second buffer pools eachcontaining about 1 μL to about 200 μL of a buffer solution. The buffersolution exhibits an affinity to non-glycosylated hemoglobin, whichfacilitates its separation from glycosylated hemoglobin. Anelectrophoresis strip is positioned within the microchannel andstructured to receive at least a portion of the hemolysate. Theelectrophoresis strip has first and second ends positioned in the firstand second buffer pools so as to be at least partially saturated withthe buffer solution in each buffer pool. A first electrode is connectedto the housing and exposed to the buffer solution in the first bufferpool. A second electrode is connected to the housing and exposed to thebuffer solution in the second buffer pool. The first and secondelectrodes generate an electric field across the electrophoresis strip.A sample loading port extends through the housing to the microchanneland provides access to a portion of the electrophoresis strip configuredto receive hemolysate of the blood sample. The application of anelectric field to the first and second electrodes induce migration andseparation of one or more bands of hemoglobin types in the sampledelivered to the electrophoresis strip through the sample loading port.A portion of the housing is optically transparent for visualizing theone or more bands of migrated and separated hemoglobin types on theelectrophoresis strip.

In some embodiments, the cartridge includes a first wall, which delimitsthe first buffer pool, and a second wall, which delimits the secondbuffer pool. The first and second walls extend the entire width of themicrochannel to prevent the buffer solution from flowing out of eachbuffer pool. At least one first restricting member extends into thefirst buffer pool and at least one second restricting member extendsinto the second buffer pool.

In other embodiments, the electrophoresis strip has a lengthsubstantially equal to a distance between the at least one firstrestricting member and the at least one second restricting member suchthat the first and second restricting members prevent longitudinalmovement of the electrophoresis strip relative to the housing. A firstelectrode connected to the housing is exposed to the buffer solution inthe first buffer pool.

In other embodiments, the cartridge includes a cover secured to thehousing. The cover includes a projection aligned with the sample loadingport and engages the electrophoresis strip when the cover is connectedto the housing for preventing movement of the electrophoresis stripduring delivery of the blood sample to the electrophoresis strip. Theapplication of an electric field to the first and second electrodesinduces migration and separation of hemoglobin in the blood sampledelivered to the electrophoresis strip through the sample loading port.

In other embodiments, the diagnostic system includes an electrophoresisband detection module structured to detect through the opticallytransparent portion of the housing the one or more bands of hemoglobintypes on the electrophoresis strip caused by the applied electric fieldand to generate band detection data based on the one or more bands ofmigrated and separated hemoglobin types.

In still other embodiments, the diagnostic system includes a processorthat receives and analyzes the band detection data to determine one ormore band characteristics for each of the one or more bands ofhemoglobin types and generate diagnostic results based on the one ormore band characteristics.

Other embodiments described herein relate to a diagnostic system foridentification and quantification of glycosylated hemoglobin (HbA1c) andnon-glycosylated hemoglobin (HbA) in a blood sample. The diagnosticsystem includes a cartridge, an electrophoresis band detection module,and a processor. The cartridge includes a housing that has amicrochannel structured to receive a hemolysate of a blood sample. Themicrochannel extends between first and second buffer pools eachcontaining about 1 μL to about 200 μL of a buffer solution. The buffersolution exhibits an affinity to non-glycosylated hemoglobin, whichfacilitates its separation from glycosylated hemoglobin. Anelectrophoresis strip is positioned within the microchannel andstructured to receive at least a portion of a hemolysate. Theelectrophoresis strip has first and second ends positioned in the firstand second buffer pools so as to be at least partially saturated withthe buffer solution in each buffer pool. A first electrode is connectedto the housing and exposed to the buffer solution in the first bufferpool. A second electrode is connected to the housing and exposed to thebuffer solution in the second buffer pool. The first and secondelectrodes generate an electric field across the electrophoresis strip.A sample loading port extends through the housing to the microchanneland provides access to a portion of the electrophoresis strip configuredto receive hemolysate of the blood sample. The application of anelectric field to the first and second electrodes induce migration andseparation of bands of HbA1c and Hb in the sample delivered to theelectrophoresis strip through the sample loading port. A portion of thehousing is optically transparent for visualizing the one or more bandsof migrated and separated hemoglobin types on the electrophoresis strip.

The electrophoresis band detection module is structured to detectthrough the optically transparent portion of the housing the HbA1c andHbA bands on the electrophoresis strip caused by the applied electricfield and to generate band detection data based on the HbA1c and HbAbands; and

The processor receives and analyzes the band detection data to determineone or more band characteristics for each of the HbA1c and HbA bands andgenerate diagnostic results indicative of HbA1c and HbA quantity in theblood sample. In some embodiments, the processor can be configured todiagnose whether the subject has or is at risk of diabetes.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 (A-B) illustrate (A) schematic view of diagnostic system inaccordance with an embodiment described herein and (B) an exploded viewof an example of a cartridge electrophoresis device.

FIG. 2 is a bottom view of the device of FIG. 1 .

FIG. 3 is a front view of an indicating member of the device of FIG. 1 .

FIG. 4 is a side view of the device of FIG. 1 and an enlarged portionthereof.

FIG. 5 is a section view of the device of FIG. 2 taken along line 5-5.

FIG. 6 is another side view of the device of FIG. 1 and an enlargedportion thereof.

FIG. 7 is a schematic illustration of the device of FIG. 1 in use.

FIGS. 8A-8E illustrate another example cartridge electrophoresis device.

FIGS. 9A-9B illustrate images showing time lapse photos and schematicillustrations of a electrophoresis test.

FIGS. 10 (A-B) illustrate schema showing (A) a method of using thediagnostic system described herein; and (B) a web-based image processingdata flow and results comparison.

FIGS. 11 (A-E) illustrate a HemeChip diagnostic system for point-of-care(POC) detection and quantification of hemoglobin variants. (A) Detaileddesign of HemeChip fabricated using injection molding. (B) HemeChip iscompact, single-use, portable, and works on the principles of celluloseacetate electrophoresis. (C) Different Hb types are separated from astamped blood sample, forming distinct bands on the cellulose acetatestrip, based on their net charge in a precisely controlled electricalfield in HemeChip. (D) For affinity cellulose acetate electrophoresis,the glycosylated Hb in the stamped sample separates from thenon-glycosylated Hb, the non-glycosylated portion has larger mobilitydue to its affinity to the buffer and thus will travel further towardsthe anode. (E) The HemeChip system is packaged inside a rugged point ofcare (POC) device with an embedded GPS designed for use in clinics aswell as remote settings.

FIGS. 12 (A-B) illustrate identification and quantification ofglycosylated hemoglobin (HbA1c) and non-glycosylated hemoglobin (HbA) inblood. Two samples from normal subjects were tested using affinitycellulose acetate electrophoresis. (A) Peak identification andquantification results show 5.2% glycosylated and 94.8% non-glycosylatedHb for subject #875. (B) Peak identification and quantification resultsshow 2.7% glycosylated and 97.3% nonglycosylated Hb for subject #017.(dashed rectangles indicate sample application locations).

DETAILED DESCRIPTION

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity but also plural entities and also includes thegeneral class of which a specific example may be used for illustration.The terminology herein is used to describe specific embodiments of theinvention, but their usage does not delimit the invention, except asoutlined in the claims.

The term “microchannels” as used herein refer to pathways through amedium (e.g., silicon) that allow for movement of liquids and gasses.Microchannels thus can connect other components, i.e., keep components“in liquid communication.” While it is not intended that the presentinvention be limited by precise dimensions of the channels, illustrativeranges for channels are as follows: the channels can be between 0.35 and100 μm in depth (preferably 50 μm) and between 50 and 1000 μm in width(preferably 400 μm). Channel length can be between 4 mm and 100 mm, orabout 27 mm. An “electrophoresis channel” is a channel substantiallyfilled with a material (e.g., cellulose acetate paper) that aids in thedifferential migration of biological substances (e.g., for example wholecells, proteins, lipids, nucleic acids). In particular, anelectrophoresis channel may aid in the differential migration of bloodcells based upon mutations in their respective hemoglobin content.

The term “microfabricated”, “micromachined” and/or “micromanufactured”as used herein, means to build, construct, assemble or create a deviceon a small scale (e.g., where components have micron size dimensions) ormicroscale. In one embodiment, electrophoresis devices aremicrofabricated (“microfabricated electrophoresis device”) in about themillimeter to centimeter size range.

The term “polymer” refers to a substance formed from two or moremolecules of the same substance. Examples of a polymer are gels,crosslinked gels and polyacrylamide gels. Polymers may also be linearpolymers. In a linear polymer the molecules align predominately inchains parallel or nearly parallel to each other. In a non-linearpolymer the parallel alignment of molecules is not required.

The term “electrode” as used herein, refers to an electric conductorthrough which an electric current enters or leaves.

The term “channel spacer” as used herein, refers to a solid substratecapable of supporting lithographic etching. A channel spacer maycomprise one, or more, microchannels and is sealed from the outsideenvironment using dual adhesive films between a top cap and a bottomcap, respectively.

The term “suspected of having”, as used herein, refers a medicalcondition or set of medical conditions (e.g., preliminary symptoms)exhibited by a patient that is insufficient to provide a differentialdiagnosis. Nonetheless, the exhibited condition(s) would justify furthertesting (e.g., autoantibody testing) to obtain further information onwhich to base a diagnosis.

The term “at risk of” as used herein, refers to a medical condition orset of medical conditions exhibited by a patient which may predisposethe patient to a particular disease or affliction. For example, theseconditions may result from influences that include, but are not limitedto, behavioral, emotional, chemical, biochemical, or environmentalinfluences.

The term “symptom”, as used herein, refers to any subjective orobjective evidence of disease or physical disturbance observed by thepatient. For example, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting.

The term “disease” or “medical condition”, as used herein, refers to anyimpairment of the normal state of the living animal or plant body or oneof its parts that interrupts or modifies the performance of the vitalfunctions. Typically manifested by distinguishing signs and symptoms, itis usually a response to: i) environmental factors (as malnutrition,industrial hazards, or climate); ii) specific infective agents (asworms, bacteria, or viruses); iii) inherent defects of the organism (asgenetic anomalies); and/or iv) combinations of these factors.

The term “patient” or “subject”, as used herein, is a human or animaland need not be hospitalized. For example, out-patients, persons innursing homes are “patients.” A patient may comprise any age of a humanor non-human animal and therefore includes both adult and juveniles(i.e., children). It is not intended that the term “patient” connote aneed for medical treatment, therefore, a patient may voluntarily orinvoluntarily be part of experimentation whether clinical or in supportof basic science studies.

The term “derived from” as used herein, refers to the source of acompound or sample. In one respect, a compound or sample may be derivedfrom an organism or particular species.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). A biological sample may comprise a cell,tissue extract, body fluid, chromosomes or extrachromosomal elementsisolated from a cell, genomic DNA (in solution or bound to a solidsupport such as for Southern blot analysis), RNA (in solution or boundto a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

Embodiments described herein relate to a diagnostic system andelectrophoresis device for detecting and/or measuring hemoglobinvariants types in blood of subject, and particularly relates to acartridge for a point-of-care diagnostic system that includes acartridge electrophoresis device for measuring hemoglobin (Hb) types,such as HbA1c. In some embodiments, the diagnostic system can be used tomeasure HbA1c levels to determine glucose levels in a subject having orsuspected of having diabetes.

FIG. 1A is a schematic illustration of a point-of-care blood diagnosticsystem 10 in accordance an embodiment described herein. A point-of-carediagnostic system includes devices that are physically located at thesite at which patients are tested and sometimes treated to provide quickresults and highly effective treatment. Point-of-care devices canprovide information and help in diagnosing patient disorders while thepatient is present with potentially immediate referral and/or treatment.Unlike gold standard laboratory-based blood testing for disorders, thedisclosed point-of-care devices enable diagnosis close to the patientwhile maintaining high sensitivity and accuracy aiding efficient andeffective early treatment of the disorder and/or infection.

The diagnostic system 10 includes a cartridge electrophoresis device 12for performing electrophoresis analysis on a sample and a reader 14 thatcan interface with the cartridge 14 to perform electrophoresis, analyzethe electrophoresis, and optionally convey and/or display the result toa user of the system 10.

Referring to FIGS. 1B-3 , an example cartridge electrophoresis device 20includes a housing 22, an indicating member 80, and a cover 60. Thehousing 22 has a generally rectangular shape and extends along acenterline 24 from a first end 26 to a second end 28. A wall 29 of thehousing 22 defines a recessed channel 30, i.e., microchannel, whichextends between the first and second ends 26, 28. The microchannel 30can be between about 0.35 and 100 μm in depth (preferably 50 μm) andbetween about 50 and 1000 μm in width (preferably 400 μm). Themicrochannel 30 length along the centerline 24 can be between 4 mm and100 mm (preferably 27 mm).

The microchannel 30 is constructed to receive an electrophoresis stripsieving medium that aids in the differential migration of biologicalsubstances, such as whole cells, proteins, lipids, and nucleic acids.More specifically, the electrophoresis strip in the microchannel 30 isconfigured to suppress convective mixing of the fluid phase throughwhich electrophoresis takes place and contributes to molecular sieving.In one example, the electrophoresis strip can constitute celluloseacetate paper. The electrophoresis channel 30 can aid in thedifferential migration of hemoglobin variants or types from a hemolysateof blood of a subject.

The wall 29 also helps to define first and second buffer pools 32, 34located at opposite ends of the channel 30. More specifically, the firstbuffer pool 32 is positioned at or adjacent to the first end 26 of thehousing 22. The second buffer pool 34 is positioned at or adjacent tothe second end 28 of the housing.

A first opening 36 extends through the bottom of the housing 22 into thefirst buffer pool 32. A second opening 38 extends through the bottom ofthe housing 22 into the second buffer pool 34. As shown, the openings36, 38 are circular. Alternatively, the openings 36, 38 can have anyround or polygonal shape. In any case, an electrode 50 is positioned inthe first opening 36 and exposed to the first buffer pool 32. Anelectrode 52 is positioned in the second opening 38 and exposed to thesecond buffer pool 34. The electrodes 50, 52 can be made from aconductive material, e.g., steel, 300 stainless steel, graphite and/orcarbon.

A wall 40 is positioned within the microchannel 30 and helps define theboundary of the first buffer pool 32. The wall 40 spans the entire widthof the microchannel 30 perpendicular to the centerline 24. A pair ofrestricting members 46 extends from the wall 29 of the housing 22towards the centerline 24. The restricting members 46 extend parallel tothe wall 40 and are positioned closer to the first end 26 than the wall.The restricting members 46 are spaced from one another and spaced fromthe centerline 24. A gap or space 54 is formed between the restrictingmembers 46 and the wall 40.

A wall 42 is positioned within the microchannel 30 and helps define theboundary of the second buffer pool 34. The wall 42 spans the entirewidth of the microchannel 30 perpendicular to the centerline 24. A pairof restricting members 48 extends from the wall 29 of the housing 22towards the centerline 24. The restricting members 48 extend parallel tothe wall 42 and are positioned closer to the second end 28 than the wall42. The restricting members 48 are spaced from one another and spacedfrom the centerline 24. A gap or space 56 is formed between therestricting members 48 and the wall 42.

An opening 90 extends through the bottom (as shown) of the housing 22into the microchannel 30 and between the walls 40, 42. The opening 90can have any shape but regardless is used as a sample loading port bywhich blood samples can be injected or otherwise supplied to themicrochannel 30, as will be described.

The indicating member 80 is elongated and includes a base 82 and a pairof legs 84 extending from the base. The indicating member 80 can beformed from the electrophoresis strip. Optionally, the electrophoresisstrip 80 can be secured to or embedded in a hard, conductive material,e.g., metal, as indicated generally in phantom at 83 in FIGS. 1-2 . Inany case, the indicating member 80 is generally rectangular with thelegs 84 extending at an angle, e.g., perpendicular, from the base 82.Consequently, the indicating member 80 can have a U-shaped construction.

A pair of electrode indications 88 a, 88 b is provided at opposite endsof the base 82. As shown, the indication 88 a is a negative (−) terminalindication and the indication 88 b is a positive (+) terminalindication. The terminal designations could, however, be reversed.

A series of test indications 86 can be provided along the base 82between the electrode indications 88 a, 88 b and parallel to the lengthof the microchannel 30. Depending on the blood test to be performed, thetest indications 86 can be symmetrically or asymmetrically spaced fromone another along the base 82. The test indications 86 can belongitudinally aligned with one another or misaligned. The testindications 86 can have the same dimensions or different dimensions fromone another. In one example, the test indications 86 are colored bandsindicative of the basic types of hemoglobin, e.g., normal hemoglobin(HbA₀), fetal hemoglobin (HbF), sickle hemoglobin (HbS), hemoglobin C(HbC or HbA₂), and non-glycosylated hemoblobin (HbA), and glycosylatedhemoglobin (HbA1c).

The cover 60 is shaped similarly to the housing 22 and extends from afirst end 61 to a second end 63. The cover 60 is generally rectangularand configured to be secured to the housing 22. The cover 60 can, forexample, form a snap-fit connection with the housing 22. In any case,the cover 60 cooperates with housing 22 to define and enclose themicrochannel 30.

A first opening 62 extends through the first end 61 and a second opening66 extends through the second end 63. A first recess 64 is formed in theunderside of the cover 60 and extends to the opening 64. A second recess68 is formed in the underside of the cover 60 and extends to the opening66.

The underside of the cover 60 further includes a plurality of supportmembers 74 positioned between the openings 62, 66. As shown, the supportmembers 74 are rectangular projections extending parallel to one anotherand perpendicular to the length of the cover 22.

A portion 69 of the housing 22 between the ends 26, 28 is transparent oroptically clear to define an optical window that allows light to passinto and/or through a portion of the microchannel 30, e.g., the testindications 86 on the indicating member 80 when positioned within themicrochannel. The ability to pass light can be a necessary step duringanalysis of a patient sample within the cartridge 20. The optical window69 can be a material and/or construction that necessarily or desirablyalters light entering the optical window 69 as a part of the analysis ofthe patient sample within, such as collimating, filtering, and/orpolarizing the light that passes through the optical window 69.Alternatively, the optical window 69 can be transparent or translucent,or can be an opening within the housing 22 of the cartridge 20. Thecartridge 20 can include a reflector (not shown) opposite the opticalwindow 69 that reflects the incoming light back through the opticalwindow 69 or through another optical window, or can include a furtheroptical window opposite the light entry window to allow light to passthrough the cartridge 20.

To this end, the portion or optical window 69 can optionally be providedwith a hydrophilic coating to prevent spotting or hazing on the portion69. The cover 60 and the housing 22 can both be formed from hard,durable materials, such as a plastic, polymer and/or glass.

When the device is assembled, the electrodes 50, 52 are positioned inthe openings 36, 38 and extend into each buffer pool 32, 34. The legs 84of the indicating member 80 are inserted into the gaps 54, 56 at eachend 26, 28 of the housing 22 such that the indicating member 80 extendsparallel to/along the housing centerline 24 within or adjacent to themicrochannel 30. The restricting members 46, 48 and walls 40, 42 arelongitudinally spaced from one another in a manner that prevents orlimits longitudinal movement of the indicating member 80 relative to thehousing 22. More specifically, the indicating member 80—in particularthe electrophoresis strip, e.g., cellulose acetate paper—has a lengthsubstantially equal to the longitudinal distance between the restrictingmembers 46, 48 such that the indicating member abuts the restrictingmembers to prevent relative longitudinal movement therebetween.

The first buffer pool 32 and the second buffer pool 34 each receive abuffer solution 51, 53 that at least partially saturates the indicatingmember 80 extending into the respective pool. The buffer solution 51, 53can exhibit an affinity to non-glycosylated hemoglobin, facilitate itsseparation from glycosylated hemoglobin, and thus be used for HbA1Ctesting.

In some embodiments, the buffer solution can be mildly acidic, forexample, a pH of about 4.5 to about 6.7, (e.g., pH 6.4), and include asulfated polysaccharide. The sulfated polysaccharide can bind to orexhibit an affinity to non-glycosylated hemoglobin. The sulfatedpolysaccharide is not particularly limited, and a known sulfatedpolysaccharide can be used. Specific examples include compounds forintroducing a sulfate group to a neutral polysaccharide, such ascellulose, dextran, agarose, mannan or starch, or a derivative thereof,and salts of thereof; chondroitin sulfate; dextran sulfate; heparin;heparan; fucoidan; and the like. In certain embodiments, the sulfatedpolysaccharide can include dextran sulfate.

The buffer solution can also include organic acids such as citric acid,succinic acid, tartaric acid, and malic acid and salts thereof; aminoacids such as glycine, taurine and arginine; inorganic acids, such ashydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boricacid and acetic acid, and salts thereof; and the like. Optionally, agenerally used additive may be added to the above-mentioned buffersolution. Examples thereof include surfactants, various polymers,hydrophilic low-molecular-weight compounds, and the like.

By way of example, the buffer solution can include 33 mmol citrate, 2μmol dextran sulfate, and 8 μmol disodium EDTA per liter, at a pH of6.4. The buffer pools 32, 34 can each receive about 1 μL to about 200 μLof the respective buffer solution 51, 53.

The cover 60 is secured to the housing 22 to confine the indicatormember 80 within the microchannel 30. The opening 62 in the cover 60 isaligned with the electrode 50 within the first buffer pool 32. Thenegative electrode indication 88 a is generally positioned between theopening 62 and the electrode 50. The opening 66 in the cover 60 isaligned with the electrode 52 within the second buffer pool 34. Thenegative electrode indication 88 b is generally positioned between theopening 66 and the electrode 52.

When the cover 60 is secured to the housing 22 the optical window 69 ofthe housing is aligned with the indicating member 80 such that all thetest indications 86 on the electrophoresis strip are visible through theoptical window 69. In other words, the support members 74 on the cover60 do not visually obstruct the test indications 86 through the opticalwindow 69.

Referring to FIG. 5 , an electrode 94 is inserted through the opening 62in the cover, into the first buffer pool 32, and into contact with theelectrode 50. An electrode 96 is inserted through the opening 66 in thecover, into the second buffer pool 34, and into contact with theelectrode 52. The electrodes 94, 96 are electrically connected to apower supply 98. The electrodes 50, 52, 94, 96, buffer solutions 51, 53and indicating member 80 cooperate to form an electrical circuit throughthe cartridge electrophoresis device 20.

The power supply 98 is capable of generating an electric field of about1V to about 400V. In some instances, the voltage applied to thecartridge electrophoresis device 20 by the electrodes 94, 96 does notexceed 250V. Regardless, an electric field is generated across theelectrophoresis strip of the indicating member 80 effective to promotemigration of hemoglobin variants in a blood sample along theelectrophoresis strip.

A patient sample, such as patient blood sample, can be provided in thecartridge 20 and on the electrophoresis strip using a sample applicator92. The sample applicator 92 provides a more precise and/or controlleddeposition of the sample onto the electrophoresis strip. Additionally,the patient sample can include added compounds/components, such as oneor more markers. The added compounds/components can assist with theelectrophoresis process and/or assist with interpreting theelectrophoresis results.

For example, the one or more markers can have known migrations ratesand/or distances for a given applied voltage and/or voltage applicationtime. Alternatively, these markers can normalize the results of theelectrophoresis process by having migration rates relative to thesample, thereby reducing the effects of sample-to-sample variability.These markers can assist with evaluating the resultant banding of thepatient sample.

A sample applicator 92 is filled with hemolysate of a blood sample BSand inserted into the loading port 90 on the underside of the housing22. The hemolysate of the blood sample BS introduced into the loadingport 90 can be, for example, less than about 10 μL. The cartridgeelectrophoresis device 20 can therefore be microengineered and capableof processing a small volume, e.g., a finger or heel prick volume.

In any case, the sample applicator 92 is urged in the direction Dtowards the indicating member 80. The loading port 90 therefore helps toguide the sample applicator 92 towards a desired location on theindicating member 80. Since the indicating member 80 is formed from theelectrophoresis strip, the sample applicator 92 can be inserted to thestrip and release the sample BS therein to the left [as shown in FIG. 6] of all the test indications 86.

The loading port 90 is aligned with and extends towards one of thesupport structures 74. As a result, the support members 74—especiallythe leftmost support member—acts as a reaction surface to the indicatingmember 80 as the sample applicator 92 extends into the electrophoresisstrip and deposits the sample therein. The support member 74 therebyprevents movement of the indicating member 80 away from the movingsample applicator 92. This helps prevent deformation or distortion ofthe indicating member 80 and helps the user release the sample in theproper location along the indicating member.

Once the sample BS is released in position, the sample applicator 92 iswithdrawn (in a direction opposite D) from the electrophoresis device20. Capillary action by the electrophoresis strip 80 can maintain thesample BS in position. The power supply 98 is actuated/turned on, whichsupplies current to the buffer pools 32, 34 and indicating member 80 asdescribed, which establishes a continuous electrical path through thedevice 20 via the buffer pools 32, 34 and cellulose acetate paper 80 incontact therewith.

In this example, forming the indicating member 80 out of celluloseacetate paper allows the indicating member to also act as the sievingmedium during the electrophoresis process. To this end, hemoglobin inthe hemolysate of the blood sample BS are moved through the indicatingmember 80 and towards the positive electrode 52 in the directionindicated by A (FIG. 7 ).

With the patient sample in place, a voltage is applied using theelectrodes, causing hemoglobin variant types to migrate across theelectrophoresis strip over a defined time. The various hemoglobin typeswill separate into bands due to the applied voltage and the physical andelectrical properties of the various hemoblobin types. One or all of theapplied voltage, current and the application time can be predeterminedor preset based on the various parameters of the electrophoresis testingbeing performed. Alternatively, one or more of the voltage, current andapplication times can be variable and based on the banding of thepatient sample or an added compound/component therein. For example, themovement of a marker added to the patient sample can be monitored as themarker moves across the electrophoresis strip. That is,imaging/monitoring of the electrophoresis testing, and/or the markersthereon, can be performed in a continuous or timed interval mannerduring the testing process. For example, images of the electrophoresisprocess can be continuously captured, such as by a video imagingprocess, or the images can be captured at regular intervals based ontime and/or the distance one or more bands have traveled. Once themarker has reached a predetermined location across the electrophoresisstrip, the test can be terminated with the removal of the appliedvoltage.

After a predetermined time, the power supply 98 is turned off and thehemoglobin variants are frozen in their respective positions along thelength of the indicating member 80 and relative to the various testindications 86. Depending on the distribution of the hemoglobin 86 adiagnosis regarding the sample BS can be made. To this end, thetransparent portion 69 of the housing 22 allows the reader to simplyvisualize the distribution of hemoglobin relative to the testindications 86 on the indicating member 80. Consequently, a diagnosisregarding the sample BS can quickly be made. In the example shown, theuser can identify different hemoglobin distributions within thehemolysate of the blood sample BS and readily diagnose any deficienciesor hemoglobin-related conditions.

The configuration of the cartridge electrophoresis device 20 isadvantageous for several reasons. First, as noted, the supportstructures 74 provided on the cover 60 help prevent movement of theindicating member 80 while the sample BS is injected/provided into thecellulose acetate paper forming the indicating member. Second, the walls40, 42 and associated restricting members 46, 48 each help prevent orlimit relative movement between the indicating member 80 and the housing22 during loading and operation of the device 20.

Moreover, the wall 40 also helps to prevent the buffer solution 51within the first buffer pool 32 from leaking into the microchannel 30via capillary action. Similarly, the wall 42 helps to prevent the buffersolution 53 within the second buffer pool 34 from leaking into themicrochannel 30 via capillary action. The walls 40, 42 help ensurecurrent flow through the device 20 is continuous and helps the devicemaintain a substantially constant pH during operation.

Additionally, embedding the electrodes 50, 52 within the bottom of thebuffer pools 32, 34 helps to ensure a consistent supply of electricfield through the cellulose acetate paper on the indicating member 80,even when/if either buffer solution 51, 53 begins to evaporate.

The reader 14 can include a housing (not shown) that surrounds andencloses some portion or all of the reader components. The housing ofthe reader 14 is constructed of materials, which may involve a suitablyrobust construction such that the reader 14 is rugged and portable.Alternatively, the reader 14 can be designed and/or constructed for usein a permanent or semi-permanent location, such as in a clinic orlaboratory.

The housing of the reader 14 includes a cartridge interface thatinteracts with and/or engages the cartridge 12 for analysis of a patientsample. The cartridge interface can be a slot that is shaped to receivethe cartridge 12. Alternative designs and/or structures of cartridgeinterfaces can be used with the reader 14.

The reader 14 can include an electrophoresis module 15 that caninterface with the cartridge 12 to perform the electrophoresis test. Theelectrophoresis module 15, alone or in conjunction with processingcircuitry, can control the electrophoresis test, includingvoltage/current application time and/or level. The electrophoresismodule can supply electrical power from the power supply 98 to thecartridge 12, or electrophoresis strip, directly, to establish thenecessary voltage across the electrophoresis strip for testing. Thevoltage can be applied at a higher level to increase the speed of thetesting, however, the increased speed can cause decreased band fidelity,which can increase the difficulty and error of the band analysis andevaluation. A lower applied voltage can increase band fidelity but canlengthen the required testing time. Alternatively, the electrophoresismodule 15 can vary the applied voltage or current, while maintaining theother stable, to achieve a desired or required level of band fidelityand testing speed. For example, an initial test to identify a patientcondition can be carried out at a higher level voltage level to speedthe test and a subsequent test to quantify the condition can be carriedout a lower voltage level to generate clearer or more accurate results.

An electrophoresis band detection module 16, alone or in conjunctionwith the electrophoresis module 15, can capture, analyze and/or evaluatethe electrophoresis test results and/or any other band detectioncharacteristic(s) related to or otherwise based on the electrophoresistest results. The electrophoresis band detection module 16 can includean imaging device, such as a digital image sensor, to capture an imageof the electrophoresis strip and the banding thereon at the conclusionof the electrophoresis test. Using the captured image data, each of thebands can be associated with one or more compounds/components of thepatient blood sample and the proportions of each can be determined.

The reader 14 can also include an output 17 that includes one or morevisual and/or audible outputs although in other examples the output isdata and does not include visual and/or audible outputs. The output 17communicates information regarding the status of the reader 14, theresults of analysis of a patient sample, instructions regarding use ofthe reader 14 and/or other information to a user or other computingdevice. The output 17 can include a display, such as a screen, such as atouchscreen, lights, and/or other visual indicators. The touchscreenused to display information, such as analysis results, to the user canalso be used by a user to input to the reader 14. Alternative interfacescan be included on and/or connected to the reader 14, such as a keyboardand/or mouse. Additionally, user devices, such as a cellphone or tablet,can be connected to the reader 14 to provide an interface portal throughwhich a user can interact with the reader 14. The audible output 17 caninclude a speaker, buzzer, or other audible indicators. The output 17can be output through an external device, such as a computer, speaker,or mobile device connected physically and/or wirelessly to the reader14. The output 17 can output data, including the collected analysis dataand/or interpretative data indicative of the presence or absence of adisorder, condition, infection and/or disease within the patient and/orthe patient sample. An example can include the identification andproportions of the various hemoglobin types within the patient sample.The interpretive data output can be based on the analysis data collectedand processed by the processing circuitry of the reader 14.

The reader can further include a sample processing module 18. The sampleprocessing module 18 can receive inputs from the electrophoresis banddetection module 16. Based on the received band detection data thesample processing module 16 can determine at least a characteristic ofthe patient sample, such as a disease or condition, an identity of thevarious compounds/components of the patient sample and quantification ofthe various compounds/components of the patient sample. The sampleprocessing module 18 can output the identification and proportions ofthe compounds/components, and/or other various data based on theanalysis of the patient sample. For example, the sample processingmodule 18, using the band detection data from the electrophoresis banddetection module 16, can identify and quantify the various hemoglobintypes of the patient sample. The output from the sample processingmodule 18 can be transmitted through the output 17 of the reader 14 ortransmitted to an external device and/or system, such as a computer,mobile device, and remote server or database.

The sample processing module 18 can analyze the patient sample todetermine a hemoglobin characteristic, such as a hemoglobin affectingdisease and/or condition, based on the data from various components,elements and/or systems of the reader 14. The results of the analysiscan be output from the sample processing module 18 to the output 17 toconvey the information to a user or other.

Referring to FIGS. 9 (A-B), another example of a cartridgeelectrophoresis device or 200 can include a housing having first andsecond buffer ports, a sample loading port, and first and secondelectrodes. The housing also includes a microchannel that extends from afirst end to a second end of the housing. The microchannel contains anelectrophoresis strip (e.g., cellulose acetate paper) that is at leastpartially saturated with a buffer solution that exhibits an affinity tonon-glycosylated hemoglobin, which facilitates its separation fromglycosylated hemoglobin, and can thus be used for HbA1C testing. Thefirst buffer port and the second buffer extend, respectively, throughthe first end and second of the housing to the microchannel andelectrophoresis strip. The first buffer port and the second buffer portare capable of receiving the buffer solution that at least partiallysaturates the electrophoresis strip.

The sample loading port can receive hemolysate of a blood sample andextends through the first end of the housing to the microchannel andcellulose acetate paper. The first electrode and the second electrodecan generate an electric field across the electrophoresis effective topromote migration of hemoglobin variants in the hemolysate of the bloodsample along the cellulose acetate paper. The first electrode and secondelectrode can extend, respectively, through the first buffer port andthe second port to the electrophoresis strip.

In some embodiments, the housing can include a top cap, a bottom cap,and a channel spacer interposed between the top cap and the bottom cap.The channel spacer can define the channel in the housing. The top cap,bottom cap, and channel spacer can be formed from at least one of glassor plastic.

In some embodiments, the diagnostic system can further include a readerthat includes an imaging system for visualizing and quantifyinghemoglobin variant band migration along the electrophoresis strip forblood samples introduced into the sample loading port. The housing caninclude a viewing area for visualizing the cellulose acetate paper andhemoglobin variant migration.

The first electrode and the second electrode can be connected to a powersupply. The power supply can generate an electric field of about 1V toabout 400V. In some embodiments, the voltage applied to the cartridge bythe electrodes does not exceed 250V.

The cartridge can be microengineered and be capable of processing asmall volume (e.g., for example, a fingerprick volume or a heelprickvolume).

The sample can be a blood sample that can be optionally treated, ifnecessary or desired, for analysis. The treatment of the blood samplecan include diluting the blood sample, which can be done by mixing thecollected blood sample with a dilutant, such as deionized water or otherfluid that dilutes the blood sample. The dilutant can alter theviscosity of the blood sample, the opacity or translucence of the bloodsample, or otherwise prepare the blood sample for analysis using thereader. Preferably, the dilutant does not impact the resulting analysisof the blood sample and/or assists with preparing the blood sample foranalysis. This can include lysing the cells of the blood sample torelease the various cellular components for electrophoresis analysis bythe reader. Lysing agents can include fluids, such as water or variouschemicals, and powders. Additionally, mechanical lysing can be used,such as by sonication, maceration and/or filtering, to achieve adequatelysing of the cells of the blood sample in preparation for analysis ofthe sample.

One or more markers can be added to the blood sample. The added markerscan assist with visualizing the completed electrophoresis results. Forexample, a marker that moves at the same relative rate as a hemoglobintype due at a predetermined applied voltage can be added. The markerwill move with the hemoglobin type containing portion of the bloodsample across the electrophoresis strip in response to the appliedvoltage. The marker can have a color, or other optical properties thatmakes visualizing the marker easier. Since the marker moves with therelative to a specific hemoglobin type, the easier to visualize markercan make it easier to determine the distance the hemoglobin type hasmoved across the electrophoresis strip in response to the appliedvoltage.

In other embodiments, the sample introduced into the sample loading portcan be less than 10 μL. The buffer solution can include alkalinetris/Borate/EDTA buffer solution. The first electrode and the secondelectrode can include graphite or carbon electrodes.

In other embodiments, the imaging system can include a mobile phoneimaging system to visualize and quantify hemoglobin variant migration.For example, as shown in FIG. 10 , the mobile phone imaging system caninclude a mobile telephone that is used to image hemoglobin variantmigration and a software application that recognizes and quantifies thehemoglobin band types and thicknesses to make a diagnostic decision. Thehemoglobin band types can include hemoglobin types C/A, S, F, A0, A1,and A1c.

In some embodiments, the diagnostic system can be used to diagnosewhether the subject has hemoglobin variants HbAA, HbSS, HbSA, HbSC,HbA2, HbA1, and HbA1c. In other embodiments, the diagnostic system canbe used to diagnose whether the subject has or an increased risk ofdiabetes.

In some embodiments, the diagnostic system can be used in a method wherehemolysate of a blood sample from a subject is introduced into thesample loading port. The blood sample includes hemoglobin. Hemoglobinbands formed on the cellulose acetate paper are then imaged with theimaging system to determine hemoglobin phenotype for the subject. Thehemoglobin phenotype can selected from the group consisting of HbAA,HbSA, HbSS, HbSC, HbA2, HbA1, and HbA1c.

In some embodiments, the cartridge comprises biomedical grade polymethyl methacrylate (PMMA, McMaster-Carr) substrates and a double sidedadhesive film (DSA)(3M Company), which have been shown to bebiocompatible and non-cytotoxic in biomedical and clinical applications.Cartridges may be fabricated using a micromachining platform (e.g.,X-660 Laser, Universal Laser Systems) to create a variety of structuresincluding, but not limited to, inlet ports, outlet ports, sample ports,microfluidic channels, reaction chambers, and/or electrophoresischannels. (FIG. 8A) Microfluidic channel dimensions may be controlled towithin 10 μm. In other embodiments, the diagnostic system allows rapidmanual assembly and is disposable (e.g., for example, a single usecartridge) to prevent potential cross-contamination between patients.

One advantage of the diagnostic system described herein, andparticularly, the cartridge electrophoresis device, is that it issuitable for mass-production which provides efficiency in point-of-caretechnologies. The diagnostic system can provide a low cost screen testfor monitoring glucose levels and diabetes in a subject. It is mobileand easy-to-use; it can be performed by anyone after a short (30 minute)training. The diagnostic system described herein can integrate with amobile device (e.g., IPhone, IPod) to produce objective and quantitativeresults. If necessary, cartridge electrophoresis devices and/or theircomponents may be sterilized (e.g., by UV light) and assembled insterile laminar flow hood. Sterile biomedical grade silicon tubing(Tygon Biopharm Plus) may be integrated to the cartridge electrophoresisdevices and cartridge electrophoresis devices may be sealed to preventany leakage. Further, tubing allows simple connection to otherplatforms, such as in vitro culture systems for additional analyses ifneeded.

In other embodiments, a mobile imaging and quantification algorithm canbe integrated into the diagnostic system and/or reader. The algorithmcan achieve reliable and repeatable test results for data collected inall resource settings of the diagnostic system.

FIG. 10A is an example analysis method 300 identifying and quantifyingglycosylated hemoglobin (HbA1c) and non-glycosylated hemoglobin (Hba).The analysis of a patient sample, which is patient blood in thisexample, is performed to determine average blood glucose concentrationover a period of two months preceding the taking of a blood sample. Theexample method of FIG. 10A can be performed using a reader andcartridge, such as the example shown in FIG. 1 . The reader can includeone or more systems and/or elements to analyze, quantify, identifyand/or otherwise determine HbA1c characteristics of a patient samplethat can be indicative of the presence of diabetes in the patient.

An initial step 302 of the method 300 can include the collection of apatient sample for analysis, in this example, a blood sample.

At 304, a buffer can be added to the electrophoresis strip inpreparation for the electrophoresis testing of the collected bloodsample. The buffer solution can exhibit an affinity to non-glycosylatedhemoglobin, facilitate its separation from glycosylated hemoglobin, andthus be used for HbA1C testing.

In some embodiments, the buffer solution can be mildly acidic, forexample, a pH of about 4.5 to about 6.7, (e.g., pH 6.4) and include asulfated polysaccharide. The sulfated polysaccharide can bind to orexhibit an affinity to non-glycosylated hemoglobin. The sulfatedpolysaccharide is not particularly limited, and a known sulfatedpolysaccharide can be used. Specific examples include compounds forintroducing a sulfate group to a neutral polysaccharide, such ascellulose, dextran, agarose, mannan or starch, or a derivative thereof,and salts of thereof; chondroitin sulfate; dextran sulfate; heparin;heparan; fucoidan; and the like. In certain embodiments, the sulfatedpolysaccharide can include dextran sulfate.

The buffer solution can also include organic acids such as citric acid,succinic acid, tartaric acid, and malic acid and salts thereof; aminoacids such as glycine, taurine and arginine; inorganic acids, such ashydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boricacid and acetic acid, and salts thereof; and the like. Optionally, agenerally used additive may be added to the above-mentioned buffersolution. Examples thereof include surfactants, various polymers,hydrophilic low-molecular-weight compounds, and the like.

By way of example, the buffer solution can include 33 mmol citrate, 2μmol dextran sulfate, and 8 μmol disodium EDTA per liter, at a pH of6.4.

The collected blood sample 302 can then be treated at step 306, ifnecessary or desired, for analysis. The treatment of the blood samplecan include diluting the blood sample, which can be done by mixing thecollected blood sample with a dilutant, such as deionized water or otherfluid that dilutes the blood sample. The dilutant can alter theviscosity of the blood sample, the opacity or translucence of the bloodsample, or otherwise prepare the blood sample for analysis using thereader. Preferably, the dilutant does not impact the resulting analysisof the blood sample and/or assists with preparing the blood sample foranalysis. This can include lysing the cells of the blood sample torelease the various cellular components for electrophoresis analysis bythe reader. Lysing agents can include fluids, such as water or variouschemicals, and powders. Additionally, mechanical lysing can be used,such as by sonication, maceration and/or filtering, to achieve adequatelysing of the cells of the blood sample in preparation for analysis ofthe sample.

At 308, one or more markers can be optionally added to the blood sample.The added markers can assist with visualizing the completedelectrophoresis results. For example, a marker that moves at the samerelative rate as a hemoglobin type due at a predetermined appliedvoltage can be added. The marker will move with the hemoglobin typecontaining portion of the blood sample across the electrophoresis stripin response to the applied voltage. The marker can have a color, orother optical properties that makes visualizing the marker easier. Sincethe marker moves with the relative to a specific hemoglobin type, theeasier to visualize marker can make it easier to determine the distancethe hemoglobin type has moved across the electrophoresis strip inresponse to the applied voltage.

At 310 the blood sample can be deposited onto the electrophoresis stripin a controlled manner, preferably applied in a “line” perpendicular tothe length of the electrophoresis strip. The controlled manner ofdeposition can include controlling the amount of blood sample deposited,the area across which the blood sample is deposited, the shape of thearea across which the blood sample is deposited and/or other depositioncharacteristics. One or more systems and/or components of the readerand/or cartridge can be used to deposit the blood sample in thecontrolled manner onto the electrophoresis strip.

With the blood sample deposited onto the electrophoresis strip, avoltage can be applied across the electrophoresis strip at 312 to causethe separation of the blood sample into various bands of glycosylatedhemoglobin and non-glycosylated hemoglobin. The voltage or current canbe applied at a predetermined level or series of levels and for anamount of time. As discussed previously, the application time of thevoltage can be predetermined or based on the movement of one or morebands of the patient sample, measurement of an electrical parameter suchas resistance or an added compound/component. A higher applied voltagecan cause the bands to move across the electrophoresis strip at agreater speed, however, the band shape can be distorted making theinterpretation of the banding difficult. A lower applied voltage canincrease band fidelity but can take a longer time to perform therequisite testing. The applied voltage can be selected to optimizetesting efficiency while maintaining a desired or minimum fidelitylevel. Further, the applied voltage can be varied during testing, suchas applying a higher voltage initially and then applying a lowervoltage. The varied application of the voltage can cause the initialband separation and movement and the later applied lower voltage canassist with increasing the fidelity of the resultant banding pattern.Additionally, varying voltages and/or currents can be applied during theelectrophoresis process in response to a measurement of the bands formedby the blood and/or the band or bands formed by the markers in apredetermined ratio, to maintain a constant rate of travel of the markerband or a portion thereof.

After completion of the electrophoresis process, the electrophoresisstrip can be optionally stained at 314. Staining the electrophoresisstrip and the bands thereon can assist with the analysis and/orevaluation of the banding. For example, a stain for hemoglobin can beused to stain the bands to assist with determining a position of thebands across the electrophoresis strip. The cartridge and/or reader caninclude the stain and the required systems/components for applying thestain to the electrophoresis strip. Alternatively, a user can stain theelectrophoresis strip before band analysis. Alternatively or inaddition, a short high voltage can be applied at the end of the testessentially burning the hemoglobin bands and making them visuallypersistent. The high voltage may also reduce the risk of viablepathogens.

At 316, the electrophoresis strip can be optically analyzed, includingimaging the electrophoresis strip and the bands thereon. Theelectrophoresis strip can be imaged using one or more light sourcesemitting one or more spectrums of light. Multiple images of theelectrophoresis strip can be captured in various lighting conditions inorder to assist with analyzing/evaluating the bands. The image capturecan be accomplished using one or more imaging sensors, such as a digitalimaging sensor and can be performed throughout the testing process or atthe conclusion of the test. The captured image(s) can be processed toevaluate and/or analyze the electrophoresis test results.

At 318, the final location and characteristics of the bands can becalculated. The calculation can determine the distance each of the bandstraveled, due to the applied voltage during testing, from the initialblood sample placement on the electrophoresis strip as well as the areaof the bands. Along with the distance of travel, a speed of travel ofeach band can be calculated based on the elapsed voltage applicationtime and the distance traveled. Using the identity, area, and locationof each of the bands, the various components/compounds of the initialblood sample, and their proportions, can be determined.

As part of the analysis of the electrophoresis tests, the bands formedduring the testing can be identified and quantified at 320.Identification of the bands can include associating one or morecompounds/components of the initial blood sample with each of the bandsof the electrophoresis. For example, identifying the bands can includeassociating each of the bands with a hemoglobin type. The identificationof the bands can be assisted by markers that were previously added tothe blood sample prior to the electrophoresis testing. The markers canbe selected so that their final position along the electrophoresis testaligns with one or more of the compounds/components of interest in theblood sample. Alternatively, the marker can be selected to beinterspersed between two bands so assist with differentiating the bandsfor identification.

Once the analysis of the blood sample is complete, the results can beoutput. The output of the results can include the identified andquantified HbA1c relative to non-glycosylated hemoglobin, which can beindicative of diabetes. The output can be displayed or relayed to theuser in a visual output, such as on a display, auditory such as by aspeaker, or other manner. This can include transmitting the outputresults to an external device, such as a computer, through a wired orwireless connection or communication protocol, such as by a Bluetoothconnection.

Referring to FIG. 10B, in some embodiments, imaging of theelectrophoresis cartridge and data analysis may be performed using areader that includes mobile or portable computer interfacable imagingsystem or application to enhance reliability and reproducibility ofblood analyses. The imaging system can include a CMOS camera used toimage, for example, hemoglobin variant migration and a softwareapplication that recognizes and quantifies the hemoglobin band types andthicknesses to make a screening decision.

To this end, an image processing algorithm can initially recognize amicrofluidic channel by using exemplary sample ports as positionmarkers. Then, red (R) pixel values may be extracted from a color imageand normalized with respect to background. Red pixel intensityhistograms may be plotted automatically along the channel, therebydetermining the positions of highest intensity (FIG. 10B)

The application segments, counts, and quantifies the bands thatcorrespond to different Hb types, and hence different Hb disorders onelectrophoresis strip. For example, Hb1A and/or Hb1Ac positions can bedetermined for each sample using histogram plots, and the resultsdisplayed on a screen. Graphical user interface includes essentialfeatures, including fiducial markers for self-calibration that guide theuser to properly align the camera field-of-view. The application caninput date, location, and a unique patient identifier.

Example Materials and Methods HemeChip Materials

A HemeChip for diagnosis of hemoglobin disorders, including diabetes,was fabricated with multiple layer lamination of PMMA encompassing asingle strip of cellulose acetate paper (FIG. 8A). In particular, poly(methyl methacrylate) (PMMA) sheets of 1.5 mm thickness were purchasedfrom McMaster-Carr (Elmhurst, Ill.), and 1/32″ thick PMMA sheets werepurchased from ePlastics (San Diego, Calif.). 3M optically clear doublesided adhesive (DSA) (Type 8142) was purchased from iTapeStore (ScotchPlains, N.J.).

Cellulose acetate membranes were purchased from Apacor and distributedby VWR International LLC (Radnor, Pa.). A 300V power supply waspurchased from VWR International LLC (Model 302, Radnor, Pa.). Ponceau SStain, Hemoglobin AFSC control, and Super Z micro-applicator werepurchased from Helena Laboratories (Beaumont, Tex.). Acetic acid glacialwas purchased from Fisher Scientific (Waltham, Mass.). Graphiteelectrodes (0.9 mm) were purchased from Amazon (Seattle, Wash.). Black⅛″ diameter dots were purchased from Mark-It (Tonawanda, N.Y.).

HemeChip Fabrication and Assembly

The DSA was used to assemble 5 layers of PMMA into a laminated, compactdesign (5 cm×2 cm×6 cm) capable of being carried in a pocket (FIG. 8B).We used the VersaLASER system (Universal Laser Systems Inc., Scottsdale,Ariz.), a laser micromachining system, for making individual PMMA layersas well as the DSA to attach the layers. The top and bottom layer of theHemeChip is made of 1/32″ thick PMMA sheet, and the rest of the layersare made of 1.5 mm thick PMMA sheet. The DSA has a thickness of 50 μm.The cellulose acetate paper for the experiments is also cut (39 mm×9 mm)using the VersaLASER system. We have also fabricated a sample loadingunit for the HemeChip using a similar manufacturing method with PMMA.The unit was compared to manual stamping by hand through analysis of thecross sectional area and repeatability of the blood sample application.

The CAD designs for the HemeChip were drawn using the CorelDRAW Suite X6(Corel Corporation, Ottawa, Ontario) and SolidWorks 3D CAD (Waltham,Mass.). The designs were exported to the interface for VersaLASER systemfor making those layers. The cutting power for the VersaLASER system wasprepped by setting the “vector cutting” from the intensity adjustmentfor the laser. The PMMA sheets and the DSA were cut with a setting of45% (min: −50%, max: 50%) for the “vector cutting”. We used a setting of−40% for cutting the cellulose acetate paper. A non-through cut at theboth ends of the plastic back of the cellulose paper was created forbending the paper (FIG. 8C). The bending facilitates the buffer solutionand paper contact for the electrophoresis. The non-through cuts werecreated at 3.0 mm off the ends using a setting of 50% (min: −50%, max:50%) of “Raster” setting at the intensity adjustment for the laser.

Blood Preparation

Under Institutional Review Board (IRB) approval, discarded andde-identified patient blood samples were obtained from UniversityHospital's Hematology and Oncology Division (Cleveland, Ohio). Bloodsamples were collected into Vacutainer tubes containing EDTAanticoagulants (BD, Franklin Lakes, N.J.). Whole blood samples werestored standing at 10° C. and left to separate into plasma andhematocrit via gravity. The hematocrit of each sample was mixed withdeionized water in a 1:5 ratio and placed on an ice block for 15 minutesto lyse the red blood cells. Prepared samples were stored in individualsealed microtubes at 10° C. and mixed gently before use. Samples wereused and stored up to two weeks from the date received. Alternatively,whole blood samples can be lysed on the chip with a 1% Saponin+TBEbuffer solution.

HemeChip Analysis

The cellulose acetate paper was soaked with 40 μL of buffer solutionthat includes 33 mmol citrate, 2 μmol dextran sulfate, and 8 μmoldisodium EDTA per liter, at a pH of 6.4 via pipette through theHemeChip's sample loading port until fully saturated by capillaryaction. Excess buffer was left to dry or redistribute through the paperfor 5 minutes. Less than 1 μL of prepared blood sample was stamped ontothe paper using a micro-applicator through the sample loading port.Approximately 200 μL of buffer was pipetted into each buffer port.Graphite electrodes (1 inch length) were placed vertically into thebuffer ports. The HemeChip was run at a constant voltage of 250V and maxcurrent of 5 mA for 8 minutes using a compact power supply. Optionally,Ponceau S stain (25 μL) was pipetted on to the paper and left to soakfor 5 minutes. A 5% acetic acid wash was used to remove the stain untilthe hemoglobin bands were visible and the paper returned to its originalwhite color. Four black ⅛″ diameter dots were placed at each corner foruse with the mobile and web-based image processing software.

Image Processing

A Nikon D3200 camera with a 40 mm f/2.8G AF-S DX Micro NIKKOR lens(Tokyo, Japan) was used to capture close up pictures of each HemeChip.Images were processed using ImageJ version 1.48 for Windows with noadditional plugins. In each image, only the paper was cropped and usedfor analysis. The Subtract Background feature was used to apply a“rolling ball” algorithm with a radius of 25 pixels to remove smoothcontinuous background noise from the paper. The Plot Profile, SurfacePlot, and Gel Plot tools were used to visualize and quantify thehemoglobin bands.

The Plot Profile tool provided the relative pixel intensities along thepaper and were used to identify the peaks corresponding to each type ofhemoglobin band. The area under each peak was calculated using the GelPlot tools and represented the relative hemoglobin percentages (see forexample FIG. 10 ). The area of each peak was outlined using thevalley-to-valley method commonly used in gas chromatography. 3D Surfaceprofiles of hemoglobin bands were obtained with the Surface Plot tool.Band distances were calculated in MATLAB to identify the coordinates ofeach peak on the profile plot. These were converted from pixels to mmusing the HemeChip length-to-pixel ratio obtained from ImageJ. The sameprocedure was used to quantify the hemoglobin results from the standardbenchtop electrophoresis setup.

Web-Based Image Processing and Quantification

Image processing algorithm: In this example the image processingalgorithm is developed using MATLAB software and shown generally at 300in FIG. 10 . Briefly, the algorithm first reads the color image, detectsthe reference points and calibrates the image dimensions of the chip andthe channel. Then the algorithm identifies the changes in red, blue andgreen values for each pixel along a reference line placed in the middleof the channel. This identification leads to detection of the peakvalues, which correspond to the reddish areas in the channel. Once thereddish areas are determined, the area of each area and the displacementfrom the start point is calculated. The area and the distance are usedto determine the type of hemoglobin disease.

In order to have an image analysis system that is independent from themobile operating systems, we designed the image-processing modulecompatible with cloud computing resources. As a result, any mobiledevice, which has a web browser and Internet connection, can be used totake image from HemeChip, transfer image to cloud computing servers foranalysis and receive/display the results on the web browser.

We used MATLAB Compiler SDK™ (Software Development Kit) to produce a.dll file from the MATLAB code to process in .NET framework. Theproduced .dll file and Bootstrap framework were used to develop Asp.Netproject. First, the webpage converts the image of HemeChip into mwArrayobject and transfers it to .NET library that runs the MATLAB code in thebackground. Then the output of the function produces another mwArrayobject and transfers back to the webpage for display. We are planning toincrease the time efficiency of the image analysis by removing ASP.NETlayer and integrating a fully scalable cloud computing system. In thisdesign image-processing request will be conveyed to queuing service.Queuing service will distribute processing requests to BackgroundWorkers in order to execute the requests and scalability will beautomatically performed based on the amount of requests. It is alsopossible to develop a mobile device application instead of running theanalysis tool on the web browser. Having an application on the mobiledevice allows controlling camera features and taking calibrated HemeChipimages.

Statistical Analysis

Band traveling distances for different hemoglobin types werestatistically assessed (Minitab 16 software, Minitab Inc., StateCollege, Pa.) using one way Analysis of Variance (ANOVA) test. Thecorrelation and agreement between the measured hemoglobin concentrationsfor HemeChip and HPLC were evaluated using Pearson-product-momentcorrelation coefficient and Bland-Altman analysis. Limits of agreementin the Bland-Altman analysis were defined as the meant different ±1.96times the standard deviation of the differences. Statisticalsignificance was set at 95% confidence level for all tests (p<0.05).Receiver-operating curves were utilized to assess differentiation ofdifferent hemoglobin phenotypes based on their traveling distances inthe HemeChip. Sensitivity was calculated as #true positives/(#truepositives+#false negatives) and specificity was calculates as #truenegatives/(#true negatives+#false positives). HemeChip data obtained inthis study is reported as mean±standard deviation. Error bars in thefigures represent the standard deviation.

Results

HemeChip was developed as the first miniaturized fully integratedsingle-use cartridge based microchip electrophoresis device for thedetection and quantification of hemoglobin variants. HemeChip technologyoffers a timely, original and innovative solution, leveraging a novelengineering approach, to POC diagnosis of hemoglobinopathies (FIGS.11A-E). HemeChip separates hemoglobin protein types in a minute volumeof blood on a piece of cellulose acetate paper that is housed in amicroengineered chip with a controlled environment and electric field(FIG. 11A-C). Differences in hemoglobin mobilities allow separation tooccur within the cellulose acetate paper. The basis of HemeChiptechnology lies in hemoglobin electrophoresis in which hemoglobin typessuch as, A (normal), S (sickle), C (hemoglobin C disease), A2 (βthal,thalassemia), Bart's, and F (fetal) have net negative charges in analkaline solution. Tris/Borate/EDTA (TBE) buffer is used to provide thenecessary ions for electrical conductivity at a pH of 8.3. The overallnegative net charges of the hemoglobins causes them to travel toward thepositive electrode when placed in an electric field (FIG. 1C).Differences in hemoglobin mobilities allow separation to occur withinthe sieving medium, cellulose acetate. HemeChip is able to evaluate allthe same variants as cellulose acetate electrophoresis, which is astandard test currently used in screening for hemoglobin disorders. Onevariation of cellulose acetate electrophoresis called affinityelectrophoresis is used for the detection of glycosylated hemoglobin.This method utilizes an acidic buffer (pH 6.4) that exhibits an affinityto non-glycosylated hemoglobin, which facilitates its separation fromglycosylated hemoglobin (FIG. 1D and FIGS. 2A&B), and can thus be usedfor A1C testing.

In our initial work aiming at validation of the cellulose acetate A1Ctesting, we ran tests with a benchtop electrophoresis system andcellulose acetate strips (Helena laboratories, Beaumont, Tex.). Twosamples from normal subjects were tested. The cellulose acetate stripswere stained and high-resolution colored images were taken. The softwareanalyses data in real-time and the image analysis process can be broadlycategorized in three stages: (i) image balance with background noisereduction, (ii) generation of intensity plot for peak detection, and(iii) calculation of area under the curve (AUC) for each detected peakto obtain relative percentages. ImageJ software was to perform the imageanalysis, in our proposed project, the image analysis procedure will beautomated with a standalone application. The peaks identified and theirrelative percentages are shown in (FIG. 12A-B).

The sample is applied at the cathode and the hemoglobins migrate to theanode under the effect of the applied electric field. For the detectionof Hemoglobin A1C, we will apply the principles of affinityelectrophoresis which utilizes a buffer containing specific chemicalscapable of interaction with specific hemoglobin types in the appliedsample. For this purpose, we will use an affinity electrophoresis bufferconsisting of 33 mmol citrate, 2 μmol dextran sulfate, and 8 μmoldisodium EDTA per liter, at a pH of 6.4. Running electrophoresis withthis buffer will take advantage of the affinity of the low-molecularmass dextran sulfate component for the non-glycosylated portion of thehemoglobin in the blood sample, which increases its mobility relative tothe glycosylated hemoglobin. This difference in mobility will allow theseparation and identification of the glycosylated hemoglobin.

Sample Preparation

Our current sample preparation steps include collection of a finger/heelprick blood sample, which is then mixed with DI water for a few minutesfor lysis. The sample preparation for A1C detection will be slightlymodified. Blood sample will have to be washed to purify the red bloodcells before lysis, which is necessary to insure that the blood serumproteins do not interfere with the detection of the hemoglobin bands.Staining solution: A staining step might be necessary in thisapplication to improve the visibility of the glycosylated hemoglobinband after separation (FIGS. 12A&B), and to improve the accuracy of thequantification results. The staining solution will be the commonly usedPonceau S. stain.

Modifications to the HemeChip Design

The current HemeChip design was scaled down from the benchtopelectrophoresis setup. The design includes a 10×40 mm cellulose acetatestrip (commercial system strips are 60×70 mm). This strip providesenough distance for the migration of all hemoglobin types detected inthe screening of hemoglobin disorders (FIG. 11B), among which HemoglobinA travels the largest distance. However, as the affinity electrophoresisemployed for this application will cause the non-glycosylated hemoglobinA to move even further, the dimensions of the cellulose acetate stripwill have to be modified to accommodate the new traveling distance. Inaddition to this change, we modified the microfluidic design of the chipto include a mechanism for introducing the stain and removing it when ithas served its purpose.

Changes to the Electrophoretic Run Parameters

The affinity electrophoresis A1C detection procedures described in theliterature for a large benchtop cellulose acetate electrophoresis systemuse 150 V run for 40 minutes. Since our HemeChip design is much smallerthan the benchtop system these parameters will be scaled down. Thevoltage and current will be scaled based on the power and currentdensities needed to achieve good electrophoretic separation combinedwith a short run time.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes, and modifications are within the skill of the artand are intended to be covered by the appended claims. All patents andpublications identified herein are incorporated by reference in theirentirety.

1. A cartridge for a diagnostic system for detecting hemoglobin in apatient sample, comprising: a microchannel extending between first andsecond buffer pools that each have a buffer solution exhibiting anaffinity to non-glycosylated hemoglobin; an electrophoresis strippositioned within the microchannel and structured to receive at least aportion of patient sample, the electrophoresis strip having first andsecond ends positioned in the first and second buffer pools,respectively, and in fluid communication with the buffer solution in therespective first and second buffer pools; a first electrode exposed tothe buffer solution in the first buffer pool; a second electrode exposedto the buffer solution in the second buffer pool, the first and secondelectrodes configured to generate an electric field across theelectrophoresis strip; and the generation of the electric field acrossthe electrophoresis strip inducing migration and separation of one ormore bands of hemoglobin types in the patient sample.
 2. The cartridgeof claim 1, further comprising a cover that includes a projectionengaged with and structured to prevent movement of the electrophoresisstrip during delivery of the patient sample to the electrophoresisstrip.
 3. The cartridge of claim 1, wherein the cartridge includes afirst wall for delimiting the first buffer pool and a second wall fordelimiting the second buffer pool, the first and second walls extendingthe entire width of the microchannel to prevent the buffer solution fromflowing by capillary action out of the first and second buffer pools,respectively.
 4. The cartridge of claim 1, further comprising a firstrestricting member extending into the first buffer pool and a secondrestricting member extending into the second buffer pool, the first andsecond restricting members preventing longitudinal movement of theelectrophoresis strip relative to the housing.
 5. The cartridge of claim4, wherein the electrophoresis strip has a length substantially equal toa distance between the first restricting member and the secondrestricting member.
 6. The cartridge of claim 5, wherein the firstrestricting member includes a pair of first restricting membersextending towards one another and parallel to the first wall, the atleast one second restricting member comprising a pair of secondrestricting members extending towards one another and parallel to thesecond wall.
 7. The cartridge of claim 1, wherein the first electrode isat least partially embedded in the first buffer pool and the secondelectrode is at least partially embedded in the second buffer pool. 8.The cartridge of claim 1, wherein the patient sample is less than 10 μL.9. The cartridge of claim 1, wherein the buffer solution is acidic andincludes a sulfated polysaccharide.
 10. The cartridge of claim 1 furthercomprising a power supply connected to the first electrode and thesecond electrode for generating the electric field across theelectrophoresis strip.
 11. The cartridge of claim 1, further comprisingan electrophoresis band detection module configured to detect the one ormore bands of hemoglobin types on the electrophoresis strip and togenerate band detection data based on the one or more bands.
 12. Thecartridge of claim 11, further comprising a processor configured to:receive the band detection data from the electrophoresis band detectionmodule, determine one or more band characteristics for each of the oneor more bands of hemoglobin types, and generate diagnostic results basedon the one or more band characteristics.
 13. The cartridge of claim 12,wherein the hemoglobin types include hemoglobin types C/A₂, S, F, A₀,A2, A1, and A1c.
 14. The diagnostic system of claim 12, wherein theprocessor is configured to determine a risk factor for diabetes based onthe one or more band characteristics.
 15. The cartridge of claim 12,wherein the electrophoresis strip defines an indicating member, theindicating member comprising a substantially U-shaped substrate in whichthe electrophoresis strip is embedded and indicia corresponding with thehemoglobin band types configured to self-calibrate the electrophoresisband detection module.
 16. The cartridge of claim 1, wherein thecartridge is configured to introduce stain to the electrophoresis strip.17-20. (canceled)
 21. The cartridge of claim 1, wherein the first andsecond ends of the electrophoresis strip are partially submerged in thebuffer solution of the respective first and second buffer pools.
 22. Thecartridge of claim 1, further comprising an optically transparent windowthrough which the one or more bands of hemoglobin types on theelectrophoresis strip are optically detectable.
 23. The cartridge ofclaim 1, wherein the first and second buffer pools each have 1-200 μL ofthe buffer solution.
 24. The cartridge of claim 1, wherein the patientsample includes a hemolysate of patient blood.